In Situ Assembly of Transmembrane Proteins from Expressed and Synthetic Components in Giant Unilamellar Vesicles.

Reconstituting functional transmembrane (TM) proteins into model membranes is challenging due to the difficulty of expressing hydrophobic TM domains, which often require stabilizing detergents that can perturb protein structure and function. Recent model systems solve this problem by linking the soluble domains of membrane proteins to lipids, using noncovalent conjugation. Herein, we test an alternative solution involving the in vitro assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic giant unilamellar vesicle (GUV) membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. We show that the extracellular domain of programmed cell death protein 1, a mammalian immune checkpoint receptor, retains its ligand-binding function at a membrane interface after ligation to a synthetic TM peptide in GUVs, facilitating the study of receptor-ligand interactions.

Microbial contamination and its associations with major ions in shallow groundwater along coastal Tamil Nadu.

Microbes in groundwater play a key role in determining the drinking water quality of the water. The study aims to interpret the sources of microbes in groundwater and its relationship to geochemistry. The study was carried out by collecting groundwater samples and analyzed to obtain various cations and anions, where HCO3-, Cl- and NO3- found to be higher than permissible limits in few samples. Microbial analysis, like total coliform (TC), total viable counts (TVC), fecal coliforms (FC), Vibrio cholera (V. cholerae) and total Streptococci (T. streptococci) were analyzed, and the observations reveal that most of the samples were found to be above the permissible limits adopted by EU, BIS, WHO and USEPA standards. Correlation analysis shows good correlation between Mg2+-HCO3-, K+-NO3-, TVC- V. cholerae and T. streptococci-FC. Major ions like Mg+, K+, NO3, Ca2+ and PO4 along with TS and FC were identified to control the geochemical and microbial activities in the region. The magnesium hardness in the groundwater is inferred to influence the TVC and V. cholerae. The mixing of effluents from different sources reflected the association of Cl with TC. Population of microbes T. streptococci and FC was mainly associated with Ca and Cl content in groundwater, depicting the role of electron acceptors and donors. The sources of the microbial population were observed with respect to the land use pattern and the spatial distribution of hydrogeochemical factors in the region. The study inferred that highest microbial activity in the observed in the residential areas, cultivated regions and around the landfill sites due to the leaching of sewage water and fertilizers runoff into groundwater. The concentrations of ions and microbes were found to be above the permissible limits of drinking water quality standards. This may lead to the deterioration in the health of particular coastal region.

Predictors of acute neurological complication following tetralogy of Fallot operation in Serdang Hospital, Malaysia.

BACKGROUND: The long waiting time for Tetralogy of Fallot (TOF) operation may potentially increase the risk of hypoxic insult. Therefore, the objective of this study is to determine the frequency of acute neurological complications following primary TOF repair and to identify the peri-operative risk factors and predictors for the neurological sequelae. METHODS: A retrospective review of the medical and surgical notes of 68 patients who underwent TOF repair in Hospital Serdang, from January 2013 to December 2017 was done. Univariate and multivariate analyses of demographics and perioperative clinical data were performed to determine the risk for the development of acute neurological complications (ANC) among these patients. RESULTS: ANC was reported in 13 cases (19.1%) with delirium being the most common manifestation (10/68, 14.7%), followed by seizures in 4 (5.9%) and abnormal movements in two patients (2.9%). Univariate analyses showed that the presence of right ventricular (RV) dysfunction, prolonged duration of inotropic support (≥7 days), prolonged duration of mechanical ventilation (≥7 days), longer length of ICU stays (≥7 days), and longer length of hospital stay (≥14 days), were significantly associated with the presence of ANCs (p<0.05). However, multivariate analyses did not show any significant association between these variables and the development of ANC (p>0.05). The predictors for the development of postoperative delirium were pre-operative oxygen saturation less than 75% (Odds Ratio, OR=16.90, 95% Confidence Interval, 95%CI:1.36, 209.71) and duration of ventilation of more than 7 days (OR=13.20, 95%CI: 1.20, 144.98). CONCLUSION: ANC following TOF repair were significantly higher in patients with RV dysfunction, in those who required a longer duration of inotropic support, mechanical ventilation, ICU and hospital stay. Low pre-operative oxygen saturation and prolonged mechanical ventilation requirement were predictors for delirium which was the commonest neurological complications observed in this study. Hence, routine screening for delirium using an objective assessment tool should be performed on these high-risk patients to enable accurate diagnosis and early intervention to improve the overall outcome of TOF surgery in this country.

A peculiar case of Morel-Lavelle lesion of upper limb.

Morel-Lavallee lesions (MLL) are post-traumatic, closed internal degloving injuries that can result in severe complications if not diagnosed early. It is conventionally seen in cases of the high energy injuries of the pelvis and lower limb. The accumulation of extravasated blood, secondary to fracture and soft tissue damage may cause internal degloving injury, skin necrosis, soft tissue damage and acute osteomyelitis. We report here the clinical and radiological features in a 32-year-old male referred from the emergency department of a tertiary hospital who had sustained high energy motor vehicle accident. On examination, there was a fluctuant, mobile, non-tender subcutaneous mass over the distal arm with suspicion of internal degloving injury. Plain radiographs showed no fractures. Ultrasound showed a fluid collection with the presence of septations and echogenic debris within the collection. Extravasation was noted between subcutaneous tissue layer and fascia at the posterolateral aspect of the arm. Wound debridement under general anaesthesia was carried out. Intraoperative findings reported a significant amount of thick serous fluid with necrotic debris. Unhealthy skin and fat layers were debrided. Underlying muscles were found to be healthy. The results of the intraoperative fluid culture and sensitivity showed no growth. Negative pressure vacuum dressing was carried out. After five cycles of vacuum dressing, the wound showed signs of healing with an improved range of motion of the elbow. Orthopaedic surgeons need to be vigilant of the possibility of MLL in the upper limb as a differential diagnosis in the management of high energy trauma.

A national audit on the utilisation and documentation of dabigatran checklist for patients initiated on dabigatran.

BACKGROUND: Direct oral anticoagulants (DOACs) especially dabigatran, have gain popularity for their efficacy, fixed dosing and favourable safety profile. A dabigatran prescribing checklist has been prepared by the Ministry of Health, Malaysia (MOH) to ensure rational and safe prescribing of dabigatran. This study therefore aimed to audit the utilization and documentation of this checklist and use of dabigatran in the government healthcare facilities. METHODS: This is a nationwide retrospective audit on the documentation of Dabigatran Prescribing and Dispensing Checklist for a period of two years from January 2013 till December 2014. Data from these Dabigatran Checklists (indication, dose, duration, renal function and adverse drug reactions encountered) were extracted by the pharmacist at MOH healthcare facilities. RESULTS: A total of 52 out of 56 (92.9%) of MOH facilities complied to usage of checklist at their centres involving a total of 582 patients of which 569 (97.7%) patients were initiated on dabigatran for the approved indications. The recommended dose of dabigatran was used correctly in 501 (99.6%) of patients. Reason for switching to DOACs use was only documented in 76.7% (131/171) of patients. The most common reason for switching from warfarin was poor INR control (n=39), history of bleeding/overwarfarinisation (n=22) and unable to attend regular INR clinic (n=21). There were 75 cases of adverse events reported. The most common adverse event reported were abdominal discomfort (n=10) followed by gum bleeding (n=9) and dizziness (n=5). CONCLUSIONS: Compliance to the dabigatran check list was high with 70% of patients prescribed the appropriate dosing.

Topical corticosteroids in clinical practice.

Topical corticosteroids are common medications prescribed for skin problems encountered in the primary care or dermatology clinic settings. As skin conditions comprise of around 20% of cases seen in primary care, this article written to guide readers, especially non-dermatologists on the appropriate potency of topical corticosteroids to be chosen for skin problems of patients and to list the side effects both local and systemic.

Case report: A case of pericardial effusion presenting to primary care.

This is a rare but interesting case of Mr. MZ who had a lifethreatening pericardial effusion presenting to the primary care clinic. Through great clinical acumen and prudent targeted investigations, diagnosis of this rare condition was reached and urgent referral made to the cardiology team which performed an emergency pericardiocentesis which proved to be life -saving. However, the hovering poor prognosis of Mr. MZ may prompt for a referral to the palliative care team to provide quality end of life care for this unfortunate patient.

Prevalence, factors influencing and knowledge about adherence to lipid-lowering therapy among hyperlipidemia patients.

BACKGROUND: Hyperlipidaemia is a significant risk factor for cardiovascular disease. However, adherence to lipidlowering therapy is often unsatisfactory due to a combination of patient factors, therapy, socio-economic and health system-related factors. AIMS: to identify the prevalence of adherence to lipidlowering therapy, the factors contributing to non-adherence and knowledge regarding hyperlipidaemia and its' treatment among Malaysian patients with hyperlipidemia. METHODS: A quantitative study using a cross-sectional survey was carried out in an urban primary care clinic in August 2015. Patients on lipid-lowering therapy for ≥ 1 year aged ≥ 18 years were selected using simple random sampling. consenting patients answered a selfadministered questionnaire (in Malay/English) which included socio-demographic profile, hyperlipidaemia profile, adherence to lipid-lowering therapy (using the Morisky Medication Adherence scale-8; score ≥ 6 taken as adherent), reasons leading to non-adherence, knowledge regarding hyperlipidaemia and its' treatment, and use of non-allopathic medicine. RESULTS: the response rate was 90.7%. the prevalence of adherence to lipid-lowering therapy was 82.4%. "the most common reasons for non-adherence was being worried about side effect of lipid-lowering agent (71.4%), followed by the need to take too many drugs in a day (61.4%) and negative influences by friends, relative and mass media (60%)". Factors associated with non-adherence include male gender, on longer duration of therapy, less frequency of follow-up, less number of follow-up clinics, taking medication at night/random timing and having lower knowledge scores. CONCLUSION: Overall the prevalence of adherence was high in patients with hyperlipidaemia. Interventions to boost adherence should target those who were identified as nonadherent.

Ruthenium photoredox-triggered phospholipid membrane formation.

As more methodologies for generating and manipulating biomimetic cellular systems are developed, opportunities arise for combining different methods to create more complex synthetic biological constructs. This necessitates an increasing need for tools to selectively trigger individual methodologies. Here we demonstrate ruthenium tris-bipyridine mediated photoredox triggering of the copper catalyzed alkyne azide cycloaddition reaction (CuAAC), resulting in the synthesis of biomimetic phospholipids in situ, and subsequent membrane assembly. The use of a ruthenium-copper electron transport chain to trigger phospholipid assembly opens up future opportunities for spatiotemporal synthesis of membranes.

Adherence of Shigella dysenteriae 1 to human colonic mucin.

The pathogenic potential of Shigella is correlated with the ability of the organism to invade and multiply within the cells of colonic epithelium. Although invasion is the ultimate event, a preceding step is adherence. Shigella dysenteriae 1 preferentially adhered to colonic mucin and not to small intestinal mucin. The pathogen showed a very strong adherence pattern to human colonic mucin when compared with guinea pig and rat mucin. The adherence pattern of S. dysenteriae 1 was not altered on preincubation with monosaccharides present in mucins, suggesting that the receptor for the pathogen is not a simple sugar. Binding of S. dysenteriae 1 to human colonic mucin was not by weak hydrophobic forces. The bacterium also adhered to glycolipids, emphasizing the role of glycoconjugates as receptors for S. dysenteriae 1.

Serum alpha-N-acetylgalactosaminidase is associated with diagnosis/prognosis of patients with squamous cell carcinoma of the uterine cervix.

Serum alpha-N-acetylgalactosaminidase (NaGalase) is responsible for the deglycosylation of vitamin D(3)-binding protein (Gc protein). The deglycosylated Gc protein cannot be converted into major macrophage-activating factor (MAF), leading to immunosuppression. NaGalase is universally detected in a variety of cancer patients, but not in healthy individuals (Cancer Res. 56 (1997) 2827-2831). However, the diagnostic/prognostic utility of NaGalase in squamous cell carcinoma (SCC) of the uterine cervix is not known. To address this issue, the serum NaGalase was quantitatively determined in 210 patients with different stages of SCC of the uterine cervix. NaGalase levels were increased with the progression of the cancer. After radiotherapy, the increased levels returned toward or to normal levels in early stages (FIGO stage I-IIB) but not in advanced stages (FIGO stage III-IV). The present study revealed that the amount of NaGalase in the patient's bloodstream reflects the tumor burden and aggressiveness of the disease. We conclude that NaGalase is an independent predictor of diagnosis/prognosis in SCC of the uterine cervix, and therefore suggest that quantitative NaGalase alteration may reflect important differences in the immunological functions of these neoplasms.

Effect of amiodarone on the membrane bound enzymes of rat intestine.

Amiodarone, a cationic amphiphile known for its clinical efficacy as an antiarrhythmic agent, unfortunately causes serious side effects. The present study was undertaken to investigate its intestinal toxicity, on oral administration, using a Wistar rat model. The relationship of drug dose and duration on intestinal toxicity was investigated. Optimum changes were observed after 21 days of AD administration at a dose of 175 mg/Kg body wt/day and this dosage was used for further studies. Histological studies revealed decreased villi and crypt size and reduction in the cellularity of lamina propria. Marked reduction in the activities of Ca(2+)-ATPase, alkaline phosphatase, disaccharidases and Na+, K(+)-ATPase was observed. The reduction in the uptake of 14C-glucose and 14C-glycine, in vivo, was correlated to the reduction in the activities of these enzymes. The reduction in the activities of the intestinal membrane bound enzymes may be attributed to altered morphology of the villi and crypts.

Enzyme-linked PNA lectin-binding assay of serum T-antigen in patients with SCC of the uterine cervix.

The expression of Thomsen-Friendenreich antigen (T-Ag) is associated with enhanced metastatic potential, poor prognosis and decreased survival rate in a variety of malignancies, and their detection and quantification can be used in serologic diagnosis. T-antigen expressions were measured by the enzyme-linked lectin assay (ELLA) with peanut agglutinin (PNA) in the sera of patients with squamous cell carcinoma (SCC) of the uterine cervix from 286 patients. This study has a sensitivity of 80%, specificity of 82% and a positive predictive value of 93%. Quantification of the T-antigen may provide useful biochemical indices for clinical assessment of the tumor spread and invasiveness of disease in SCC of the uterine cervix. Moreover, the ELLA assay is cheap, easy to perform and reproducible in the prognosis and diagnosis of SCC of the uterine cervix.

Effect of eugenol on drug-metabolizing enzymes of carbon tetrachloride-intoxicated rat liver.

The chemoprotection extended by eugenol against carbon tetrachloride (CCl4) intoxication was established by studies on drug-metabolizing phase I and phase II enzymes. An overall decrease in drug-metabolizing enzymes, namely NADPH-cytochrome c reductase, NADH-cytochrome reductase, coumarin hydroxylase, 7-ethoxy coumarin-O-deethylase, UDP-glucuronyltransferase and glutathione-S-transferase, was observed with CCl4 intoxication, with a subsequent decrease in cytochrome P450 and cytochrome b5 content. CCl4 caused a significant decrease in microsomal phospholipids and the marker enzymes glucose-6-phosphatase and 5'-nucleotidase, and an increase in thiobarbituric acid reactive substances (TBARS). Simultaneous administration of eugenol with CCl4 inhibited the accumulation of TBARS and the decrease in the microsomal phospholipids and marker enzymes. Further, the chemical onslaught imposed by CCl4 on the drug-metabolizing system was removed successfully by eugenol. Eugenol appears to act as an in vivo antioxidant and as a better inducer of phase II enzymes than phase I enzymes. It is therefore suggested that eugenol could be an interesting basic structure for drug design.

Effect of chloroquine on rat liver mitochondria.

Chloroquine causes an increase in phospholipid and a decrease in cholesterol in liver mitochondria. A significant decrease in the activities of mitochondrial inner membrane enzymes such as NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase is observed. Decrease in cytochrome contents and respiratory control ratio, shown by a decrease in state 3(+ADP) and an increase in state 4 (-ADP), implies decreased ATP synthesis following chloroquine administration. The results confirm drug-induced inhibition of mitochondrial respiration, thereby impairing availability and utilisation of energy.

Differential binding of Pseudomonas aeruginosa to normal and cystic fibrosis tracheobronchial mucins.

Pseudomonas aeruginosa infection is a leading cause of deterioration of pulmonary function in patients with cystic fibrosis (CF). The interaction of the bacterium with CF and non-CF tracheobronchial mucins was examined to understand the biochemical basis for the high susceptibility of the lungs of CF patients to infection by P.aeruginosa. The binding of radiolabelled bacteria to pure mucins in solid-phase assays was not significantly above non-specific binding to various blocking agents, such as bovine serum albumin, Tween 20, milk powder and polyvinyl pyrrolidine. Further, there was a tendency for the bacteria to be excluded from plastic wells and membranes coated with mucin. Therefore, an indirect approach involving the binding of radiolabelled P.aeruginosa to asialo GM1 ganglioside, the putative receptor for the bacteria on tracheal cells, was used to compare the interaction of CF and non-CF mucins with the bacteria. Highly purified preparations of CF mucin were consistently better inhibitors of the binding of the bacteria to asialo GM1 ganglioside than non-CF mucin preparations. In the case of the binding of a stable mucoid strain, the difference was statistically significant (P < 0.001) at all concentrations of mucin tested. For the non-mucoid strain, the difference was significant only at the higher concentrations. Of the saccharides tested similarly, sialyl lactose and the oligosaccharide portion of asialo GM1 were found to be good inhibitors. The increased binding of the bacteria to CF mucin was further confirmed by a solution binding assay in which the binding of 125I-labelled mucin to unlabelled bacteria was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiodarone--induced changes in surfactant phospholipids of rat lung.

Amiodarone HCl (AD) is a very effective antiarrhythmic drug, but its use is often associated with serious pulmonary complications. It is shown to induce lung phospholipidosis. Nevertheless, the effects of this drug on pulmonary surfactant which is composed of about 75% phospholipids and which prevents alveolar collapse is not known. Therefore, we have examined the effect of AD on the intra- and extracellular surfactant pools and on the levels of phosphatidylcholine (PC), the primary constituent of pulmonary surfactant. Male Wistar rats were fed AD (175 mg/kg) by oral gavage for three weeks. At the end of the experimental period, the rats were killed, the lungs removed and perfused, and surfactant isolated. Some lungs were prepared for ultrastructural examination. Phospholipid was assayed in the intra- and extracellular surfactant. Amiodarone produced a significant increase in both the intra- and extracellular surfactant phospholipid along with an appreciable change in the phospholipid profile. Also, the drug seemed to increase the number of lamellar inclusions in the surfactant producing type II alveolar cells. These data suggest that administration of AD leads to an increase in the lung surfactant phospholipid levels and lamellar bodies in alveolar type II cells.